DESCRIPTION For the vertebrate brain to acquire the specific connections necessary for function, the axons of millions of neurons must navigate through complex environments to find their appropriate targets. The zebrafish retinotectal neurons must navigate through complex environments to find their appropriate targets. The zebrafish retinotectal project is a well-defined system for detailed analysis of axon pathfinding in a vertebrate. Developing retinal axons in lower vertebrates exit the retina at the optic nerve head, cross the ventral midline, and then project dorsally to the contralateral optic tectum where they terminate. In ashtray, a retinotectal pathfinding mutant generated in a recent genetic screen, some retinal axons navigate successfully to the contralateral optic tectum, but many project to a genetic screen, some retinal axons navigate successfully to the contralateral optic tectum, but many project to a multitude of inappropriate targets. The ashtray defect is eye-autonomous, suggesting that the mutated gene may be involved in retinal axon responses to pathfinding cues. The four aims of this proposal are designed to gain a better understanding of the mechanism by which ashtray affects retinal axon pathfinding. If will first analyze the projections of individual ashtray retinal axons in order to determine whether they make specific or distributed pathfinding errors of individual ashtray retinal axons in order to determine whether they make specific or distributed pathfinding errors or whether inappropriate branching is responsible for the ashtray defect. Second, I will test whether correctly- and incorrectly-projecting axons arise from distinct populations of retinal ganglion cells in astray. Third, I will test whether correctly- and incorrectly-projecting axons arise from distinct populations of retinal ganglion cells in ashtray. Third, I will analyze growth cone behavior and calcium signaling in vivo in order to gain further insight into the mechanism of astray function. And fourth, I will genetically fine-map astray, test candidate genes for astray, and if necessary, find genetic function. And fourth, I will genetically fine-map ashtray, test candidate genes for ashtray, and if necessary, find genetic markers within a reasonable distance of ashtray with which to initiate a chromosome walk. The information gained from these studies should provide insight not only into the mechanism by which ashtray affects axon guidance, but also into the mechanisms of normal retinal axon pathfinding. A comprehensive understanding of normal pathfinding behavior will contribute to understanding the mechanisms by which nerves regenerate, and may illuminate the basis for human developmental disorders.